Apart from the PCR technology, immunological tests still play a major role in infection serology. By the specific determination of different-immunoglobulin classes, immunology makes it possible to analyze the stage of a disease. By determining IgM and IgG titers against certain viral antigens it becomes possible to distinguish between different infection stages, e.g., acute infections, recurrent infections, chronic/persistent infections or post-infectious disease stages. For instance, IgG molecules against the viral glycoproteins are only generated at a late stage of an infection, with cytomegalovirus (Schoppel et al. JID (1997) 175, 533-544; Eggers et al., J. Med. Virol. (2001) 63, 135-142).
A crucial aspect of a reliable specific detection of IgG and IgM in the presence of the respective other immunoglobulin class is the effective epitope concentration or effective epitope density of the detection antigen. A high effective epitope concentration means that there is a high epitope density and all epitopes are accessible for antibody binding. In contrast to this, also polypeptide aggregates have a high epitope concentration, but the effective epitope concentration is low because the epitopes are partially or completely buried or hidden and thus not accessible for antibody binding. High epitope concentrations are a precondition for specific IgM recognition whereas low epitope concentrations are a precondition for the specific recognition of IgG. In a classic sandwich IgM test, which shall detect IgM molecules against the antigen A, a multimeric antigen A is used as an immobilized capture antigen. The same multimeric antigen A carrying a reporter group is used as a detection antigen. The IgM analyte binds to the capture antigen and the detection antigen whereby the reporter group is immobilized on a solid phase (e.g., beads coated with streptavidin). In order to avoid interferences of IgG molecules directed against A, an unlabelled monomeric antigen A is added as an interference elimination agent to the test (WO 98/23955, U.S. Pat. No. 6,489,129 B1). Correspondingly, a specific IgG sandwich test (WO 98/23961, U.S. Pat. No. 6,645,732 B1) may comprise a monomeric antigen for the detection of IgG and an unlabelled multimeric antigen to avoid interferences with IgM.
The principle which allows a specific detection of IgM and IgG molecules is based on their respective molecular structures. The pentameric IgM has ten identical paratopes for antigen binding whereas the monomeric IgG has only two binding sites per molecule. The detection of IgG is based on the affinity to the analyte whereas the detection of IgM is based on the avidity. In the first case, the binding is achieved by a high affinity interaction between epitope and paratope (i.e., IgG antigen binding site); in the latter case it is achieved by a cooperative enhancement of several low affinity interactions (avidity means that the single dissociation constants are not added but multiplied, i.e., a relatively weak interaction with a kD of ˜10-5 M is increased by two independent binding events to yield a high affinity interaction with a kD of ˜10-10 M). Thus, it can be said as a rule of thumb that monomeric antigens are used for the detection of IgG and oligomeric/multimeric antigens are used for the detection of IgM.
Antigen oligomerization or multimerization may be achieved by chemical crosslinking of monomeric antigens by homo- or heterobifunctional crosslinkers. Generally, the oligo- or multimerization may be optimized by adjusting the reaction conditions (concentration of protein and crosslinker, pH, temperature, agitation rate, reaction time), which is very time-consuming and labor-intensive. Nevertheless, different degrees of crosslinking may be obtained in different batches, which require subsequent fractionation and/or calibration procedures. Further, higher degree of crosslinking normally leads to a reduction of solubility, which may lead to problems in the test performance. It is thus desired to find an improved method to provide multimeric antigens in a defined and reproducible manner.
U.S. Pat. No. 6,207,420 describes a fusion sequence comprising a carrier protein comprising an E. coli protein having a predicted solubility probability of at least 90% fused to a target heterologous peptide or protein. Preferably, the heterologous peptide or protein is normally insoluble when expressed in bacteria.
WO 03/000878 describes a fusion protein comprising at least one target polypeptide and upstream thereto at least one FKBP chaperone, which is selected from the group consisting, of FkpA, SlyD and trigger factor. The target polypeptide may be a mammalian gene product or a gene product of a mammalian pathogen.